Work in our laboratory spanning more than two decades has demonstrated that certain drugs may be attached to well-defined carrier molecules and still retain the ability to bind to the receptor site and induce biological activity. This synthetic strategy for the attachment of drugs to carriers is termed the functionalized congener approach. The carrier molecule may be many times larger than the parent drug; indeed there is practically no maximum size limitation for a fully potent analog. Unlike the prodrug approach or the immobilization of drugs for slow release, the functionalized congener approach is designed to produce analogs for which no metabolic cleavage step is necessary for activation. Moreover, the attachment of the drug to a carrier such as a peptide may result in enhanced affinity at an extracellular receptor site and an improvement in the pharmacological profile of the parent drug through energetically favorable interaction with distal sites on a receptor. The recently determined X-ray structures of the ARs follow upon a two-decade long progression of knowledge of the AR binding site(s) and other structural features, based on empirical probing of structure activity relationships (SARs) of ligands, molecular modeling, and mutagenesis analysis. Early modifications of both adenosine agonists and alkylxanthine antagonists located regions in these two ligand classes that were amenable to chemically functionalized chain extension without losing the ability to bind to the ARs. This logically suggested, long before the structure or even polypeptide composition of the ARs was known, that the putative binding sites had regions that were accessible to the external environment and therefore less demanding sterically, i.e. the functionalized chains protruded beyond the conformationally and sterically restricted binding region of the core pharmacophore. Points on the adenosine scaffold that displayed this characteristic and could be extended chemically, without a limiting length, included the N6 position for the A1AR and the C2 position for the A2AAR. In the xanthine series, such modification of the C8 position was relatively insensitive in receptor binding, and thus served as a suitable site for derivatization in AR antagonist functionalized congeners. A terminal amino group of the high affinity AR antagonist XAC (a xanthine amine congener) served as a site for generalized coupling to much larger moieties that extended into the extracellular medium, without losing AR affinity. The recently determined X-ray structures of adenosine and XAC (Dor et al., 2011) bound to a thermostabilized hA2AAR show that the terminal amino chain of XAC proved to be very flexible and not anchored to the receptor in a specific conformation. It occupied a groove formed between Tyr9 (1.35, using the universal GPCR residue identifier, Ballesteros et al. 1995) and Tyr271 (7.36), two residues that can adopt two different rotameric states depending on the ligand. Thus, the objective in the design of XAC as a functionalized congener, in which the distal amino group escapes the steric constraints of the pharmacophore binding site, was finally explained structurally. We have invented a fluorescence polarization probe MRS5346 for drug screening, which is based on a known high affinity A2AAR antagonist scaffold. Fluorescent ligands have advantages over radioligands in drug discovery. Using flow cytometry, a high affinity A3AR antagonist MRS5449 saturated the receptor with very high specific-to-nonspecific binding ratio and equilibrium binding constant of 5.15 nM. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell flow cytometry were consistent with radioligand binding in membranes. Thus, MRS5449 is a useful tool for hA3AR characterization. We have explored the application of nanotechnology to the study of GPCRs. For example, dendrimers are tree-like polymers that have multiple functional sites on the periphery for attachment of ligands. We have extensively explored tree-like polyamidoamine (PAMAM) dendrimers as nanocarriers for functionalized congeners, i.e. strategically derivatized ligands for tethering, for ARs and P2YRs. Multivalent GPCR Ligand Dendrimer (GLiDe) conjugates often display increased receptor selectivity/affinity. We have already shown that such conjugates of AR agonists, optionally bearing PEG groups for increasing water solubility, bound to the receptor and displayed antithrombotic activity (A2AAR) or cardioprotective activity (A3AR). Methods of tethering A2AAR agonists to quantum dots while retaining activity have been compared. It is becoming apparent that the receptors have higher degrees of organization, i.e. they form functionally complex aggegrates, and there is a need for pharmacological tools that address this multiplicity. Striking differences have been noted between the monomeric ligands and the multivalent dendrimer conjugates - either in the potency or selectivity of the ligand or in the kinetics of the biological effect. We recently showed that a dendrimeric adenosine derivative selective for the A3 receptor was highly protective to rat cardiac cells in culture and in an isolated heart model that were exposed to ischemic stress. The dendrimer was considerably more potent in protection than the corresponding nucleoside monomer. We have shown that a modified version of an antiasthamtic drug theophylline that might also have neuroprotective or anticancer properties, can be coupled to PAMAM dendrimeric polymers to form a multivalent display. These conjugates, which are highly potent as antagonists of adenosine receptors without cleavage of the drug, were synthesized using click chemistry, and the substitution was varied systematically to demonstrate cooperativity of binding that could indicate bridging multiple receptors. This study of antagonist conjugation is an example of how to generally improve the effectiveness or selectivity of a wide variety of small molecules that act at GPCRs.